Ketoprofen is an .alpha.-methylarylacetic acid analgesic/antiinflammatory currently available as the racemic mixture. Because the S-enantiomer has been believed to possess advantages over the R-enantiomer as an analgesic, and because S-.alpha.-methylarylacetic acid analgesic/antiinflammatory agents are generally believed superior to their R counterparts, there is extensive literature on the enantioselective production of S-ketoprofen. We have recently discovered that R-ketoprofen enjoys some previously unappreciated advantages as an analgesic and antipyretic. A process for the enantiospecific production of R-ketoprofen on a commercial scale is therefore of considerable utility and interest.
The enantioselective hydrolysis of racemic ketoprofen esters to produce S-ketoprofen is known in the art. Iriuchijima and Keiyu in an early paper [Agric. Biol. Chem. 45, 1389-1392 (1981)] disclosed the modestly selective hydrolysis of racemic ketoprofen methyl ester to S-ketoprofen with 38% enantiomeric excess (e.e) in unspecified, low yield using Mycobacterium smegmatis. Sih (European application 227078) disclosed the still modestly selective hydrolysis of racemic ketoprofen methyl ester to S-ketoprofen with 60% ee in unspecified yield using Candida cylindracea ester hydrolase. Cobbs et al. (U.S. Pat. No. 5,108,916 and PCT application WO90/15146) subsequently disclosed the more selective hydrolysis of alkyl, haloalkyl and glyceryl esters of racemic ketoprofen by ester hydrolases from Candida rugosa (formerly called Candida cylindracea) to yield S-ketoprofen in very high enantiomeric excess at 20 to 30% conversion. This was accomplished by purifying and separating the ester hydrolase isozymes from C. rugosa.
The enantioselective hydrolysis of racemic ketoprofen esters to produce R-ketoprofen has also been reported, albeit in low yield or low ee. Iriuchijima and Keiyu (op. cit.) reported that the methyl ester of ketoprofen was "hydrolyzed a little" by Aspergillis sojae to give an undisclosed ee of R-ketoprofen. Goswami (PCT application WO91/13163) disclosed the hydrolysis of racemic ketoprofen methyl ester to R-ketoprofen with 74% ee in 8% conversion by dog liver acetone powder. This is far too inefficient to provide a commercially useful process for R-ketoprofen, even if dog liver were an inexpensive reagent. Cobbs PCT application WO90/15146 appears to disclose the hydrolysis of racemic ethylene glycol ester of ketoprofen to R-ketoprofen in 42 to 64% ee by pig liver esterase and by Mucor miehei ester hydrolase.
Wu et al. [J. Am. Chem. Soc. 112, 1990-1995 (1990)] have defined a useful measure for enantioselective reactions which combines both the ee and the extent of conversion. It is termed the enantiomeric ratio E, and is defined as: ##EQU1## ee.sub.s is the enantiomeric excess of substrate (in this case the racemic ester)
ee.sub.p is the enantiomeric excess of product (in this case the R-acid) PA1 (a) reacting racemic ketoprofen with a suitable precursor to produce a choline ester of racemic ketoprofen; PA1 (b) treating the choline ester of racemic ketoprofen in water with a fungus of the species Beauveria bassiana to produce preferentially R-ketoprofen in the presence of S-enriched ketoprofen choline ester; and PA1 (c) isolating the R-ketoprofen from PA1 (a) reacting racemic ketoprofen with an activating agent to provide an activated ketoprofen; PA1 (b) reacting the activated ketoprofen with choline to provide a choline ester of racemic ketoprofen; PA1 (c) treating the choline ester of racemic ketoprofen in water with a fungus of the species Beauveria bassiana to produce a mixture consisting essentially of S-enriched ketoprofen choline ester and R-ketoprofen; PA1 (d) separating the R-ketoprofen from the S-enriched ketoprofen choline ester; and PA1 (e) hydrolyzing the S-enriched ketoprofen choline ester. PA1 (a) extracting a plurality of fractured cells of Beauveria bassiana with an aqueous PA1 (b) filtering or centrifuging the buffer to recover impure ester hydrolase in an aqueous filtrate or supernatant; and PA1 (c) contacting the supernatant with a weakly basic anion exchange resin, such as a diethylaminoethyl (DEAE) resin, to produce an aqueous solution of ester hydrolase. The ester hydrolase has an activity of greater than 100 units per milligram of protein in the solution and is capable of hydrolyzing racemic ketoprofen choline ester to R-ketoprofen in greater than 90% ee.
The enantiomeric excess is well known in the art and is defined for a resolution of ab.fwdarw.a+b as ##EQU2## The enantiomeric excess is related to the older term "optical purity" in that both are measures of the same phenomenon. The value of ee will be a number from 0 to 100, zero being racemic and 100 being pure, single enantiomer. A compound which in the past might have been called 98% optically pure is now more precisely described as 96% ee. Processes that yield products of ee less than about 80% are not generally regarded as commercially attractive. Similarly, for commercial processes a goal is to maximize the E value; E values less than 10 are undesirable. The process of Goswami to provide R-ketoprofen has a low enantiospecificity, a very low conversion, and an E value of less than 7 calculated by this method.
There is a need for a commercially useful process for R-ketoprofen. In one approach this devolves to a need for a process for the enantioselective hydrolysis of a racemic ketoprofen ester to produce R-ketoprofen by a process having an E value greater than 10.
Producing R-ketoprofen by selective hydrolysis gives rise to a second consideration: the ester from which to produce R-ketoprofen. The alkyl esters of ketoprofen are insoluble in water and are generally poor substrates for commercial enzymatic hydrolysis because multiphasic/heterogeneous reaction systems suffer from a number of drawbacks on the industrial scale. For example, scale-up and reliability problems are frequently associated with the processing of dispersions and emulsions, and continuous operation and pH control (especially in hydrolytic reactions) are difficult to achieve. Additionally, the phases must be separated before product can be recovered, and excessive interphase mass transfer resistances are often encountered. These are associated with diffusion of the poorly soluble substrate in the aqueous phase. Many of these disadvantages associated with the enzymatic resolution of water insoluble esters of chiral carboxylic esters in heterogeneous reaction systems could be minimized or eliminated if the water-solubility of the ester derivative could be substantially increased.
One approach, as shown for example in the Cobb PCT application, is the addition of surfactants to the solution broth. This appears to provide little advantage. A second approach is the use of water soluble esters. An example of this approach can be found in Dodds et al. European application 461043 which describes a preparation of the choline ester of ketoprofen and its transesterification to R-ketoprofen ethyl ester in very low ee by a protease. (Examples 18 and 20 in the Dodds application report that the transesterfication products had rotations of -0.166.degree. and -0.203.degree. respectively; pure R-ketoprofen ethyl ester has a rotation of -45.5.degree. under the conditions reported.)
Because the primary object of the present invention is to provide a commercially useful process for R-ketoprofen, there is a need for a highly efficient synthesis of a water soluble ester that can be enantioselectively hydrolyzed. There is then a need for an enantioselective hydrolysis of that ester.